Automated microdissection instrument and method for processing a biological sample

ABSTRACT

Systems and methods for automated laser microdissection are disclosed including automatic slide detection, position detection of cutting and capture lasers, focus optimization for cutting and capture lasers, energy and duration optimization for cutting and capture lasers, inspection and second phase capture and/or ablation in a quality control station and tracking information for linking substrate carrier or output microdissected regions with input sample or slide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. patent applicationSer. No. 17/516,605, filed Nov. 1, 2021. U.S. patent application Ser.No. 17/516,605 is a continuation case of Ser. No. 16/790,595, filed Feb.13, 2020, which issued as U.S. Pat. No. 11,175,203 on Nov. 16, 2021.U.S. Pat. No. 11,175,203 is a divisional application of U.S. patentapplication Ser. No. 16/208,557, filed Dec. 3, 2018, which issued asU.S. Pat. No. 10,605,706 on Mar. 31, 2020. U.S. Pat. No. 10,605,706 is adivisional application of U.S. patent application Ser. No. 15/434,200,filed Feb. 16, 2017, which issued as U.S. Pat. No. 10,156,501 on Dec.18, 2018. U.S. Pat. No. 10,156,501 is a divisional application of U.S.patent application Ser. No. 14/275,812, filed May 12, 2014. U.S. patentapplication Ser. No. 14/275,812 is a divisional application of U.S.patent application Ser. No. 11/236,045, filed Sep. 26, 2005, whichissued as U.S. Pat. No. 8,722,357 on May 13, 2014. U.S. Pat. No.8,722,357 claims priority to U.S. Provisional Patent Application No.60/664,438, filed Mar. 23, 2005, and to U.S. Provisional PatentApplication No. 60/613,038, filed Sep. 25, 2004. All applicationsidentified in this section are incorporated by reference herein, each inits entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of laser microdissection.More particularly, the invention relates to an automated lasermicrodissection instrument.

BACKGROUND

Tissue biopsies are frequently obtained for diagnostic and therapeuticpurposes. Typically a tissue biopsy sample consists of a 5 to 10 micronslice of tissue that is placed on a glass microscope slide usingtechniques well known in the field of pathology. The tissue sample willtypically consist of a variety of different types of cells. Often apathologist will desire to remove only a small portion of the tissue forfurther analysis. Before the advent of laser microdissection,pathologists would have to resort to various time-consuming andimprecise microdissection techniques to obtain a sample of the desiredregion of a biopsy. Laser microdissection provides a simple method forthe procurement of selected human cells from a heterogeneous populationcontained on a typical histopathology biopsy slide. The lasermicrodissection technique is generally described in the publishedarticle: Laser Capture Microdissection, Science, Volume 274, Number5289, Issue 8, pp 998-1001, published in 1996, incorporated herein byreference, and in the following U.S. Pat. Nos. 5,859,699; 5,985,085;6,184,973; 6,157,446; 6,215,550; 6,459,779; 6,495,195; 6,512,576;6,528,248 all herein incorporated by reference in their entirety.

Laser microdissection systems generally comprise an inverted microscopefitted with a laser. Tissue samples are mounted, typically on a standardglass slide, and a transparent thermoplastic transfer film is placedover the tissue section. This film is often manufactured containingorganic dyes that are chosen to selectively absorb in the near infraredregion of the spectrum overlapping the emission region of common AlGaAslaser diodes. When the film is exposed to the focused laser beam theexposed region is heated by the laser and melts, adhering to the tissuein the region that was exposed.

The laser melts the film in precise locations which serves to bind thefilm to a targeted cell or cells. Individual cells or clusters of cellscan be targeted by the laser, depending on the diameter of light emittedfrom the laser. Heat generated by the laser is dissipated by the film,thus limiting the damage done to the targeted cells and the componentstherein. After the targeted cells are bound to the film, they areremoved from the sample. The targeted cells are then extracted forfurther analysis. The transfer film can be mounted on a transparent capthat fits on a microcentrifuge tube to facilitate extraction.

The following invention is a new method and apparatus for lasermicrodissection that solves a number of problems of conventional lasermicrodissection.

BRIEF SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, there is provided amethod for laser microdissection. The method includes the step ofproviding a layer of biological material that is applied to the surfaceof a first substrate. A polymer layer is provided. At least one targetedportion of biological material located on the first substrate isidentified. The polymer layer is placed in juxtaposition with the firstsubstrate on the side of the biological material in the location of theat least one targeted portion of biological material. A laser source isprovided and activated so as to describe at least one closed orsubstantially closed path around the at least one targeted portion ofbiological material or directly at the least one targeted portion ofbiological material. At least one portion of biological material istransferred from the layer of biological material to the polymer layer.The polymer layer is moved to a quality control station. At least oneportion of biological material that is present on the polymer layerwhile the polymer layer is located in the quality control station isfurther identified. The at least one laser source may be activated anddirected at the at least one portion of identified biological materialthat is present on the polymer layer while the polymer layer is locatedin the quality control station.

In accordance with another aspect of the invention, there is provided amethod for automatically determining the location of a laser beamprojection on a worksurface area of a laser microdissection instrumentthat is operatively coupled to a microprocessing device and a digitalimage acquisition system containing a digital image sensor. The methodmay include the step of increasing the intensity of the laser beam. Inanother variation, the method includes emitting laser light at a levelsufficient to be detected by the digital image sensor. The increasedlight intensity of the laser beam is detected by the digital imagesensor. The pixel location of the increased light intensity on thedigital image sensor is determined and converted to a coordinatelocation corresponding to the worksurface area. The coordinate locationis assigned as the location of the laser beam from which laser cuttingor capture operations proceed.

In accordance with another aspect of the invention, there is provided amethod for optimizing the focus of a laser beam in a lasermicrodissection instrument. The method includes the step of providing alaser microdissection instrument having a worksurface. A first lasersource and laser focusing lens is disposed on a first side of theworksurface. An objective lens and image acquisition system is disposedon a second side of the worksurface. A sample is placed on theworksurface. The objective lens is focused on the sample for a clearimage of the sample acquired by the image acquisition system. The firstlaser source is activated to emit a laser beam directed at the sample.The laser beam from the first laser source is focused by moving thelaser focusing lens. The objective lens is re-focused on the sample bymoving the objective lens a distance. The laser beam from the firstlaser source is kept at the desired focus by moving the focusing lens bysubstantially the same distance that the objective lens was moved whenre-focused.

In accordance with yet another aspect of the invention, there isprovided a method for a laser microdissection instrument. The methodincludes the step of providing a first substrate having a transfer filmattached. At least one second substrate having biological material isalso provided. The second substrate with the biological material isintroduced into the laser microdissection instrument. At least onetargeted portion of biological material on the second substrate isidentified. The first substrate is placed in juxtaposition with thesecond substrate on the side of the biological material in the locationof the at least one targeted portion of biological material. A firstlaser source is provided and activated to adhere at least one region ofthe transfer film to at least one portion of biological material. Atleast one portion of biological material is transferred from the secondsubstrate to the first substrate. At least one tracking information isrecorded and associated with the first substrate.

In accordance with another aspect of the invention, there is provided adigital microscope for observing a sample. The digital microscopeincludes a worksurface for receiving a sample. The worksurfaceintersects a primary optical axis of the microscope. Asubstrate-receiving location is provided on the worksurface forreceiving a sample-bearing substrate. The worksurface includes a firstopening in the substrate-receiving location for alignment with theprimary optical axis to permit pathing of light through the firstopening in the worksurface. The digital microscope includes a digitalimage acquisition system that includes an image sensor configured toautomatically detect the presence of the substrate in thesubstrate-receiving location.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other advantages of the invention will become apparentupon reading the following detailed description and upon reference tothe drawings in which:

FIG. 1 is a depiction of a laser microdissection process shown in foursteps according to the invention;

FIG. 2 is a perspective view of the laser microdissection instrumentconnected to a computer and display according to the invention;

FIG. 3 is a perspective view of the laser microdissection instrumentwithout the housing according to the invention;

FIG. 4 is a perspective view of the worksurface of the lasermicrodissection instrument according to the invention;

FIG. 5 is a front perspective view of the cutting laser componentsmounted on the frame of the laser microdissection instrument accordingto the invention;

FIG. 6 is a rear perspective view of the fluorescence system mounted onthe frame of the laser microdissection instrument according to theinvention;

FIG. 7A is a top planar view of a biological sample with targetedportions encompassed by traces according to the invention;

FIG. 7B is a top planar view of a biological sample with targetedportions encompassed by cut paths according to the invention;

FIG. 7C is a top planar view of a biological sample with targetedportions encompassed by cut paths that are interspersed with bridgesaccording to the invention;

FIG. 8A is a top planar view of a biological sample with capture lasershots located interior of the cut paths according to the invention;

FIG. 8B is a top planar view of a biological sample with capture lasershots located in between the bridges according to the invention;

FIG. 8C is a top planar view of a biological sample with an capturelaser path that is curved across the interior of a cut path according tothe invention;

FIG. 9 is a top planar view of a biological sample with targetedportions of biological material removed according to the invention;

FIG. 10 is a side elevation view of a transfer film carrier withtargeted portions adhered thereto and separated from the remainingtissue sample according to the invention;

FIG. 11 is a top planar view of an image of a transfer film properlywetted by a capture laser according to the present invention; and

FIG. 12 is a top planar view of an image of a calibration matrix ofcapture laser spots according to the present invention.

While the invention is susceptible to various modifications andalternative forms, specific variations have been shown by way of examplein the drawings and will be described herein. However, it should beunderstood that the invention is not limited to the particular formsdisclosed. Rather, the invention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention and the various features and advantageous details thereofare explained more fully with reference to the nonlimiting embodimentsthat are illustrated in the accompanying drawings and detailed in thefollowing description. Descriptions of well known components andprocessing techniques are omitted so as not to unnecessarily obscure theinvention in detail.

The entire contents of U.S. Pat. No. 6,469,779 filed Feb. 4, 1998,entitled “Laser Capture Microdissection Device”; U.S. Pat. No.5,859,699, filed Feb. 7, 1997; U.S. Pat. No. 6,495,195, filed Feb. 14,1997; and U.S. Pat. No. 5,985,085, filed Dec. 4, 1997 are herebyexpressly incorporated by reference into the present application as iffully set forth herein.

With reference to FIG. 1 , a laser microdissection device operates tocarry out the following general steps. A tissue 10 or smear fixed on astandard microscope slide 12 by routine protocols is introduced into alaser microdissection instrument. A polymer film or transfer film 14 isprovided which is typically affixed to a solid substrate forming acarrier 16. The carrier 16 can be of any shape. One shape for thecarrier is a cap for conveniently introducing a sample into a vessel,such as a microcentrifuge tube, and sealing the vessel. The words “cap”and “carrier” are used interchangeably and it is understood by oneskilled in the art that the carrier can be of any shape even where theterm “cap” is employed.

The tissue sample 10 mounted on a substrate surface is placed adjacent atransfer film carrier cap 16 which further ensures that transfer film 14stays out of contact with the tissue 10 at this stage as shown in stepone of FIG. 1 . Alternatively, the transfer film carrier 16 can beplaced in contact with the tissue 10. Upon visualizing the tissue 10 bya microscope, a user may select a region for microdissection. Theselected section of the tissue is captured by pulsing at least oneregion of the transfer film with a low power infrared laser emitting alaser beam 18 which activates the transfer film 14 which then expandsdown into contact with the tissue 10 as shown in step two of FIG. 1 .The at least one activated region 20 of the transfer film 14 adheres tothe at least one identified portion of desired cell(s) 22 of the tissuesample. Microdissection is completed by lifting the transfer filmcarrier 16, with the desired cell(s) 22 attached to the transfer film 14surface while the surrounding tissue remains intact as shown in stepthree of FIG. 1 . Extraction and subsequent molecular analysis of thecell contents, DNA, RNA or protein, are then carried out by employingdevices and standard methods as shown in step four of FIG. 1 anddescribed in U.S. application Ser. No. 09/844,187 entitled “Lasercapture microdissection (LCM) extraction device and device carrier andmethod for LCM fluid processing” incorporated herein by reference in itsentirety.

Laser microdissection employs a polymer transfer film that is placed injuxtaposition to the tissue sample. The transfer film may or may notcontact the tissue sample. This transfer film is typically athermoplastic manufactured containing organic dyes that are chosen toselectively absorb in the near infrared region of the spectrumoverlapping the emission region of common AlGaAs infrared laser diodes.When the film is exposed to the focused laser beam the exposed region isheated by the laser and melts, adhering to the tissue in the region thatwas exposed. The film is then lifted from the tissue and the selectedportion of the tissue is removed with the film. Thermoplastic transferfilms such as a 100 micron thick ethyl vinyl acetate (EVA) filmavailable from Electroseal Corporation of Pompton Lakes, N.J. (typeE540) have been used in LCM applications. The film is chosen due to itslow melting point of about 90° C.

With reference to FIG. 2 , a laser microdissection instrument 24 isshown connected to a computer 26 with a hard drive and an LCD monitor27. The computer 26 includes the Windows operating system and basicWindows applications and is loaded with appropriate software to controlinstrument operation. The computer receives input from the user andcontrols the operation of the laser microdissection instrument 24. Thelaser microdissection instrument components are secured within a housing28 that includes an automated sliding door 30 for accessing theinstrument and inserting and removing tissue samples, slides andtransfer film carriers.

Referring now to FIG. 3 , there is shown the laser microdissectioninstrument 24 with the housing 28 removed. The laser microdissectioninstrument 24 includes a microscope 32 mounted on an assembly frame 34.The microscope 32 includes an illumination system 36, a worksurface 38,a handling system 40 and an optical system 42. The microscope frame 34carries the components of the microscope 32. The illumination system 36comprises a white light illuminator 44 and a condenser 62 mounted on theframe 34. The illumination system 36, worksurface 38 and optical system42 are arranged in an inverted transmitted-light microscope fashion suchthat the illumination system is arranged above the worksurface 38 and atleast one objective is arranged below the worksurface 38.

The worksurface 38 is also mounted on the instrument frame 34 and isadapted for receiving one or more specimens and transmitting lighttherethrough. A vacuum chuck may also be included to secure the specimenmounted on a specimen holder in position. The worksurface 38 operates asa translation stage and is automatically or manually movable in alldirections, in particular, the planar X-Y directions. The automatedtranslation stage includes a lateral translation motor and afore-and-aft translation motor to allow complete manipulation in the X-Yplane. The motors are controlled by a controller connected to thecomputer which receives input such as via a mouse cursor. A mouse cursorcan be used by an operator to trace a path on a visual display unitdepicting a live or static image of the specimen to effect movement ofthe worksurface. A sophisticated road-map imaging system for navigatingthe specimen is described in U.S. Patent Publication No. 2002-0090122which is incorporated herein by reference in its entirety.

With particular reference to FIG. 4 , there is shown the worksurface 38.The worksurface 38 includes slide locations 46 for handling multipletissue samples simultaneously. Although three slide locations 46 aredepicted, the invention is not so limited and any number of slidelocations is within the scope of the present invention. Each slidelocation 46 is designed to receive a substrate surface bearing sampletissue for microdissection. In the embodiment depicted in FIG. 4 , thesubstrate surface to be received within the slide locations 46 is astandard microscope slide. Accordingly, the slide locations areappropriately dimensioned although the invention is not so limited andany type and style of substrate surface may be adapted to fit acustomized worksurface. Each slide location 46 includes at least oneopening 48 in the worksurface 38. The worksurface 38 further includes astaging area 50 for receiving transfer film carriers 16 or capcassettes. The worksurface 38 also includes an unload station 52 forunloading caps from the slide locations 46 after microdissection. Theunload station 52 includes an unload slot 56 for receiving an unloadtray onto which caps are placed. The worksurface 38 also includes aquality control station 54. The quality control station 54 includes anopening (not shown) in the worksurface 38 that permits illumination andlaser light to pass. The quality control station 54 is designed forviewing the cap following cell capture, generating an image of the cap,and/or further ablating portions of the collected sample residing on thecap after cell capture. These features of the invention will bediscussed in greater detail below.

The handling system 40 is connected to the frame 34 and comprises a liftfork 58. The lift fork 58 is moved in and out of the work surface by atranslation motor and a lift motor operates to move the lift forkvertically. The lift fork is adapted to pick a carrier located at astaging or supply area of the worksurface and place the carrier injuxtaposition with the tissue specimen located in one of the slidelocations 46. When microdissection is completed, the lift fork isadapted to pick the carrier from juxtaposition with the specimen andplace it in the unload station 52 and/or quality control station 54where the carrier may further cap an analysis vessel. The handlingsystem also includes a visualizer filter. The visualizer filter is apiece of diffuser glass positioned above tissue sample. The light fromabove is diffused by the visualizer filter illuminating the sample fromall angles. The visualizer filter can be moved in and out of positionand is located on the lift fork. The automated handling system isdescribed in detail in U.S. Pat. No. 6,690,470 to Baer et al. and isincorporated herein by reference in its entirety.

The optical system 30 of the microscope includes several opticalelements known to a person skilled in the art to make a microscope andlaser microdissection instrument operate properly. These elements,mounted on the instrument frame, are combined to create an optical trainof optical elements for pathing light. The optical system includes butis not limited to the following optical elements: mirror(s), dichroicmirror(s), lens(es), objective, beam-diameter adjuster, cut-off filter,diffuser, condenser, eyepiece and image acquisition system such as acamera.

The optical system together with its optical elements is arranged suchthat white light from the illumination system 36 passes down toward theworksurface 38. The white light passes a dichroic mirror 60 and afocusing condenser lens 62. The white light passes through one of theopenings in the worksurface 38 along a primary optical axis and entersan objective 64 located beneath the worksurface 38. Multiple objectivesare located on an objective turret wheel 66 which is automaticallycontrolled by the computer. White light from the objective 64 is thenreflected by one or more mirrors 68 to an eyepiece (not shown) and/or acamera or image acquisition system 70. The live image captured by theimage acquisition system 70 is transmitted to the computer and displayedon a visual display unit in a software application window for the livevideo. Static images may also be captured by the image acquisitionsystem and displayed side-by-side with the live video on the visualdisplay unit in a software application window for the static roadmapimage. A cut-off filter may be located between the objective and theimage acquisition system or eyepiece. A diffuser and a beam diameteradjuster (not shown) may also be incorporated in the optical train andlocated between the dichroic mirror and the translation stage. A seriesof microscope objectives may be selectably deployed from an objectiveturret wheel 66 which is controlled by an objective wheel motor while asecond objective focus motor operates to automatically adjust the fociof objectives which have been positioned. One skilled in the art willunderstand that the optical elements may be arranged in various ways foroptimum performance.

Connected to the microscope and mounted on the instrument frame 34 iscapture laser source 72. The capture laser source is typically aninfrared (IR) laser source such as a AlGaAs laser diode having awavelength of approximately 810 nanometers. The laser diode withcollimating optics emits a beam of IR laser light that is incident uponthe dichroic mirror 60. The capture or infrared laser beam enters theoptical train at the dichroic mirror 60 and is reflected downwardthrough the focusing condenser lens 62 and/or beam diameter adjustertoward the worksurface 38. Simultaneously, the dichroic mirror 60 allowswhite light from the illumination system 36 to also pass towardworksurface resulting in the IR laser beam and the white lightillumination being superimposed along the primary optical axis. A laserfocus motor which is connected to the controller and computer operatesto control the focusing lens 62 and adjust the IR laser beam spot size.The computer 26 also delivers signals to the IR laser via the controllerto initiate IR laser pulses, adjust beam size and control IR laserpower.

The capture laser 72 operates in two modes, idle mode and pulse mode. Inidle mode, the IR laser beam path provides a visible low amplitudesignal that can be detected via the image acquisition system 70 when avisual alignment of the laser spot with a portion of tissue is desired.In pulse mode, the IR laser beam path delivers energy formicrodissection and the optical characteristics of a cut-off filterattenuate the IR laser beam path sufficiently such that substantiallynone of the energy from the IR laser beam exits through the microscope.

Suitable capture laser pulse widths are from 0 to approximately 1second, preferably from 0 to approximately 100 milliseconds, morepreferably approximately 50 milliseconds. In one variation, the spotsize of the laser at the transfer film is variable from approximately1.0 to 100 microns, from 1 to 60 microns, or from 5 to 30 microns. Fromthe standpoint of the clinical operator, the widest spot size range isthe most versatile. A lower end point in the spot size range on theorder of 5 microns is useful for transferring single cells.

Suitable lasers can be selected from a wide power range. For example, a100 milliwatt laser can be used. On the other hand, a 50 mW laser can beused. The laser can be connected to the rest of the optical subsystemwith a fiber optical coupling. Smaller spot sizes are obtainable usingdiffraction limited laser diodes and/or single mode fiber optics. Singlemode fiber allows a diffraction limited beam.

While the capture laser diode can be run in a standard mode such asTEM₀₀, other intensity profiles can be used for different types ofapplications. Further, the beam diameter could be changed with a steppedlens (not shown) placed in the lens assembly. Changing the beam diameterpermits the size of the portion of the transfer film that is activatedto be adjusted. Given a tightly focused initial condition, the beam sizecan be increased by defocusing. Given a defocused initial condition, thebeam size can be decreased by focusing. The change in focus can be infixed amounts. Furthermore, the change in focus can be obtained by meansof indents on a movable lens mounting and/or by means of optical glasssteps. In any event, increasing or decreasing the optical path length isthe effect that is needed to alter the focus of the beam, therebyaltering the spot size. For example, inserting a stepped glass prisminto the beam so the beam strikes one step tread will change the opticalpath length and alter the spot size.

Referring now to FIG. 5 , there is shown a front perspective view of acutting laser source 74 in addition to related and interconnectedcutting laser components such as a cutting laser power supply 76 and airchannel 78 installed onto the frame 34 of the instrument 24 of FIG. 3 .While FIG. 3 does not show the cutting laser source 74, it is to beunderstood that the cutting laser and its components are integrated intothe instrument 24 of FIG. 3 in the manner shown in FIG. 5 and that FIG.5 shows only the detail of the cutting laser components with otherinstrument components removed for clarity.

The cutting laser source 74 is connected to the microscope and it istypically an ultraviolet (UV) laser source 74. The UV laser source emitsa beam of laser light that is reflected by one or more mirrors anddirected into the primary optical axis. The UV laser light enters theoptical train and is reflected upward through the objective lens 64. Theobjective 64 focuses and adjusts the UV laser beam diameter. The UVlaser beam then travels toward the worksurface 38 and through one of theopenings in the worksurface and at a tissue sample residing on a slidelocated in the slide location 46 or at a captured tissue sample locatedon a cap in the quality control station 54. It is understood that theworksurface 38 is automatically moved to align particular openings inthe worksurface with the optical paths of the lasers or primary opticalaxis for the intended operation. Simultaneously, the dichroic mirror 60allows white light from the illumination system to also pass toward theworksurface 38 resulting in the UV laser beam and the white lightillumination being superimposed along the primary optical axis.Alternatively, the UV laser can be positioned above the worksurface 38and directed through the focusing lens 62 along the primary axis andtoward the specimen resting on the worksurface 38. The computer 26 alsodelivers signals to the cutting UV laser via the controller to initiateUV laser pulses, change beam diameter and control cutting UV laserpower. UV laser pulse widths and beam diameter can be changed in thesame manner as described above with respect to the IR laser source.

Focusing now on FIG. 6 , there is shown a rear perspective view of afluorescence system 80 connected to the frame 34 of the instrument 24.FIG. 6 displays the related and interconnected components of thefluorescence system 80 such as the power supply and light source 82, EPIfluorescence illuminator 84, filter wheel 86 and controller 88 installedonto the frame 34 of the instrument 24 of FIG. 3 . While FIG. 3 does notshow the fluorescence system 80, it is to be understood that thefluorescence system is integrated into the instrument 24 in the mannershown in FIG. 6 in addition to the components shown in FIG. 3 andoptionally with the components of FIG. 5 and that FIG. 6 shows only thedetail of the fluorescence system with other instrument componentsremoved for clarity purposes only.

The fluorescence system 80 is adapted for automated selection of cellsor specific regions of a sample for microdissection usingfluorescently-stained tissue samples. In image analysis, thefluorescently-labeled tissue is placed in a microdissection instrumentand with the fluorescent system, the cells are detected through ananalysis of the image formed by the microscope. Image analysis is knownin the art and is also described in detail in WO 2004/025569 which isherein incorporated by reference in its entirety.

The fluorescence system 80 includes a fluorescence excitation lightsource, for example, a xenon or mercury lamp, which emits a specificwavelength or wavelength range. The specific wavelength or wavelengthrange of a beam emitted by the light source is selected by afluorescence filter wheel 86 operated by a fluorescence filter changermotor, to excite a fluorescent system (chemical markers and opticalfiltering techniques that are known in the industry) that isincorporated in or applied to the sample to be microdissected. Thewavelength range transmitted from the excitation light source can beselected. The sample includes at least one member selected from thegroup consisting of chromophores and fluorescent dyes (synthetic ororganic), and the process of operating the instrument includesidentifying at least a portion of the sample with light that excites atleast one member, before the step of transferring a portion of thesample to the laser microdissection transfer film. The fluorescent beamcan be made coincident or coaxial with both the IR/UV laser beam pathand the white light from illuminator path. Fluorescence emitted by thesample is amplified by an objective changer 66, reflected by a camerachanger mirror and captured for live viewing by the acquisition system70 which comprises a camera. A filter wheel 86 motor operates to adjustthe fluorescent beam and the emitted fluorescent beam. Optionally theobjective changer may be implemented in the form of a wheel toaccommodate a multiplicity of objectives (five objectives, as depicted)for providing different amplifications of the fluorescent image for thecamera. A more detailed exposition of automated fluorescent lasermicrodissection is found in U.S. Pat. No. 6,690,470 which isincorporated herein by reference in its entirety.

Referring back to FIGS. 1-6 , a sample of biological material 10 to bemicrodissected is applied to a substrate such as a glass slide 12 usingroutine protocols. The substrate with the sample affixed thereto isinserted into the laser microdissection instrument 24 through automaticdoor 30 and inserted into a slide opening 48 located on the worksurface38. The instrument 24 automatically detects the presence of a slide 12when a slide is inserted into a slide opening 48 located on theworksurface 38. This automatic slide detection can be accomplished inmany ways known to one skilled in the art. For example, when a slide isinserted into a slide opening 48, a camera 70 or other sensor includedin the instrument 24 calculates a brightness differential indicative ofthe presence of a slide and an appropriate signal is registered with theuser. When the slide opening 48 is empty, the camera 70 or other sensorreads a first brightness level of the illumination light. When a slideis inserted into a slide opening 48, the camera image sensor or othersensor calculates a second brightness reading wherein the secondbrightness reading is lower than the first brightness reading andthereby, indicating that a slide has been inserted. Alternatively, whena slide is inserted into a slide opening 48 on the worksurface 38, alight path is blocked by a spring-set flange or by the slide itselfindicating the presence or absence of a slide. Upon automatic detectionof the slide 12 in the slide opening 48, the camera 70 automaticallyrecords a static image of the slide to create a roadmap image anddisplays it on the visual display unit connected to the computer in asoftware application window for the static roadmap image.

The handling system 40 is used to bring a carrier 16 with a transferfilm 14 affixed to its surface from the cap staging area 50 to the slidelocation 46 and in juxtaposition to the sample. A software interfacewindow displayed on the visual display unit depicts the worksurfacegraphic and movement of caps is effected by moving an input device suchas a mouse, clicking on a cap and dragging it onto the graphic slidelocation desired and releasing the mouse button. Software andcontrollers engage the handling system to effect movement accordingly.The worksurface 38 is automatically moved into the primary optical axissuch that a selected opening associated with a slide location 46 orquality control station 54 in the worksurface is aligned with theoptical axis and ready for microdissection. The carrier 16 is placed incontact with the sample such that the transfer film contacts thebiological material substantially across the entirety of the transferfilm surface. Alternatively, the carrier 16 is formed with standoffs 90such that a substantial portion of the transfer film 14 does not contactthe biological material 10 but remains spaced a distance from the sample10 as shown in step one of FIG. 1 . Standoffs are described in U.S.patent application Ser. No. 08/984,979 which is herein incorporated byreference in its entirety. Standoffs are structural features thatprotrude from the surface of the carrier on the side of the transferfilm to provide a spacing between the transfer film and the sample inorder to avoid transfer of unwanted friable biological material thatwould otherwise adhere to the transfer film due to electrostatic forcesand the like.

With the sample in the optical axis, the illumination system 36 isactivated shedding light on the sample 10. The white light penetratingthe sample arrives at the objective 64 and is directed to theacquisition system 70 and/or eyepiece. A live image that is captured bythe acquisition system 70 is displayed on the computer monitor 27. Also,a static image of relatively lower magnification is captured so as toprovide a roadmap image for navigating the sample space. The two imagesare displayed side-by-side to locate the user on the sample space mapand simultaneously provide a display of the local sample space having arelatively larger magnification. The operator inspects the sample bymoving the translation stage via computer inputs, controllers andappropriate software. For example, navigation of the sample space isaccomplished by tracing a path on the displayed monitor image using aninput cursor via a mouse, joystick or other input means.

Referring now to FIG. 7 , a targeted portion 92 of biological material94 is identified either manually by the operator or automaticallyemploying software for algorithmic identification of regions ofinterest. Typically, fluorescent systems are employed for assisting theautomated identification of targeted portions of biological material.Manually, the user can trace a targeted portion 92 of biologicalmaterial viewed on the display monitor by moving a mouse cursor. Eachtrace 96 defines an interior 98 and an exterior 100. The interior 98includes the targeted portion(s) and the exterior 100 of the traceincludes non-targeted biological material. One or more targeted portionsof biological material can be traced and the trace can be of any shapeand size as shown in FIG. 7 a . Various software microdissection toolsfor selecting targeted portions of biological material are available.For example, on the software interface, a user selects certainmicrodissection tools for indicating which cells the capture laser orcutting laser will capture or ablate, respectively, and operate thetools on the live or static image displayed on the computer monitor.Microdissection tools include single point dissection for targetingcells individually, line dissection for drawing a line on the image totarget a layer or line of cells, free form dissection for identifying anarea to be targeted, and exclusion dissection for deselecting a specificarea from targeting. The instrument is optionally selecting for captureonly with the capture laser as described with respect to FIG. 1 orablation with the cutting laser combined with capture with capture laserin a process known as “cut-and-capture”.

In cut-and-capture mode, the trace defines a cut line for the cuttinglaser source. After all of the targeted portions 92 have been traced theuser is prompted by the computer to commence cutting the traces with thecutting laser source 74. The user may select whether each of the tracesare to be closed or substantially closed paths for the cutting laser 74.If the user selects closed paths, the cutting laser source isautomatically directed and activated to cut along the traces at apredefined cut width 102 forming a cut path 101 as shown in FIG. 7 b .If the user selects a substantially closed path, at least one bridge 104spanning from the interior 98 to the exterior 100 will be formed suchthat the interior 98 is joined to the surrounding exterior 100biological material at the location of the bridge 104 as shown in FIG. 7c . The cut path 101 is interspersed with bridges 104 formed when the UVcutting laser beam is temporarily de-activated while moving along atrace. The bridge width 108 can be selected by the user or predeterminedby controlling software. Bridge locations may be user-defined byclicking with the mouse cursor along the trace at locations wherebridges are desired as shown by the “x” in FIG. 7 a . The user therebymanually selects any number and location of the bridges. Alternatively,the computer may automatically form a predefined number of bridges. TheUV laser is activated and the biological material is eroded along thecut path but at bridge locations, biological material remains intact.

During the cutting operation of the UV laser, the laser beam remainsstationary and the worksurface 38 serves as a cut line control unit andgenerates, during the cutting operation, a relative movement between thelaser beam and the sample. Alternatively, the cut line control unitcomprises a laser scanning device which moves the laser beam relative tothe stationary sample during cutting. In such an operation, theworksurface 38 with the sample is not displaced during cutting butremains fixed in the optical axis. The cut line results exclusively fromdeflection of the laser beam over the sample.

Typically, after the UV laser has cut the biological material along oneor more of the trace paths 96, the capture laser 72 is directed at theone or more interiors 98 of the trace paths 96. The IR capture laser 72is fired or pulsed at an interior 98 to activate the transfer film layerin the location of the interior which then adheres to the interiorportion of the biological material. If a carrier with standoffs is beingemployed, the activated transfer film bridges the distance of thestandoffs 90 to contact and adhere to the interior of biologicalmaterial. An IR laser pulse showing a location of adhesion to theinterior of biological material is shown as a circle 106 in FIG. 8 a.

The IR capture laser 72 can be fired once to create a single area ofadhesion or the IR laser can be fired more than once to create more thanone area of adhesion on any one interior portion of biological material.The single IR laser shot can be directed in the center of the interior.In another variation, the IR laser shot can be directed at the interiorof the trace but at a portion of the interior that was not targeted asdesirable biological material as shown in FIG. 8 a . In essence, ifthere is a portion of the interior which contains biological materialthat is not considered to be a choice selection or otherwise nottargeted as desirable, the IR laser can be strategically directed atsuch a location to advantageously avoid raising the temperature ofdesired biological material in the area of the IR laser shot which wouldresult from localized heating. If bridges are left by the UV lasertrace, IR laser shots shown as circles 106 on FIG. 8 b can be directedin-between the bridge locations so that such points of adhesion wouldassist in the breaking of the bridges when the carrier is lifted away.Also, the IR laser shots can be directed at or in the proximity of thebridge locations.

If the IR laser shots are delivered manually, a user can, for example,click with a mouse cursor at a location where the user desires an IRlaser shot to be located. Also, the user may select the number of IRlaser shots that are to be made by clicking with a mouse cursor morethan once.

If the IR laser shots are delivered automatically, computer software isprogrammed by the user beforehand or determined automatically to carryout one or more IR laser shots in a uniform or non-uniform pattern of IRlaser shots across the interior of a trace. Of course, a single IR lasershot as well as a strategically placed IR laser shot can also be carriedout automatically by the computer.

Furthermore, the IR laser shot is not limited to being a single pulse tocreate a single point of adhesion. Alternatively, the IR laser can befired with multiplicity or at duration to trace an IR path 110 ofadhesion of any shape within the interior as shown in FIG. 8 c . The IRlaser path of adhesion is carried out in the same manner as the UV laserpath of cutting. Either the worksurface 38 is moved to create a path orthe IR laser beam is directed across the interior with the worksurfaceremaining stationary. Basically, the number of IR laser shots, the shapeof the IR laser shots and their location are not limited and any number,pattern, location or shape of IR laser shots is within the scope of theinvention. Furthermore, the IR laser shot or shots can be fired beforethe UV laser is activated to cut the biological material.

The carrier with the transfer film will result in one or more areas ofadhesion located in the one or more interiors of the cutting laser 74cut paths. When the carrier is removed by lifting it vertically, thecarrier with its attached transfer film and at least one adheredtargeted portion of biological material is separated from the remaininglayer of biological material. If bridges were formed, those bridges aremechanically broken upon lifting the carrier to free the adheredportions of targeted biological material. What remains is un-targetedbiological material as shown in FIGS. 9 and 10 . Being adhered to thetransfer film, the targeted biological material is removed with thecarrier and available for further processing.

If the capture laser 74 shots are delivered automatically and theautomatically locate capture laser option is selected in the software,appropriate computer software is programmed to automatically detect theposition of the capture laser 74 prior to its activation. Because thecapture laser beam can be located anywhere within a predetermined area,it is advantageous to automatically determine the position of thecapture laser to provide starting coordinates from which the capturelaser beam can be accurately directed to the capture locations. In olderinstruments, the capture laser is located manually by the user. Tomanually locate the laser beam, the user moves the worksurface 38 sothat a clear laser beam spot appears in the center of the live videowindow. The user then places the point of the cursor of the computermouse for example, directly on the center of the laser beam spot andthen indicates to the computer that the laser beam has been located byright-clicking on the mouse button and selecting that the location ofthe laser has been selected. Even in manual capture laser operation,automatically locating the capture laser beam establishes a connectionbetween the cursor and the laser beam so that when the user clicks tofire or target the capture laser, the laser accurately fires on thecells that the user selected.

In the present invention, the instrument 24 automatically locates thecapture laser beam without requiring any user intervention. Automaticdetection of the capture laser beam is accomplished with appropriatecomputer software and controllers directing the system to locate thecapture laser. First, in one variation, the background illumination suchas the white light illuminator 44 is automatically turned off. This stepis useful in situations where the capture laser targeting beam is tooweak to be seen by the user such as when the capture laser beam isoperating in idle mode or if the laser beam intensity is on a lowsetting. Therefore, it is useful to turn off the background illuminationor alternatively, increase the laser beam intensity. In anothervariation, the laser is set at a level sufficient to be detected by thedigital image sensor. This level is generally selected to be below thepower level that melts the polymer film. After turning off thebackground illumination and/or increasing the laser beam intensity, thelaser beam spot is detected by the camera as a bright spot relative tothe neighboring areas. The exact location of the bright spot and laserbeam location is calculated from brightness levels detected by thedigital camera image sensor of the acquisition system 70. Given aparticular magnification, the pixel coordinates of the beam spotlocation are translated to planar coordinates for establishing a zeroedlocation from which the worksurface or the laser beam is preciselydirected to areas of interest. Prior to determining the exact locationof the laser beam, in one variation, the intensity of the capture laseris increased or fired so that a bright spot is more easily detected. Thetransfer film is not activated when the laser is fired to locate thebeam because the light intensity is set to a low level. After the laserbeam is located the background illumination light is turned back on ifit was previously turned off. The same procedure described above may beperformed for locating the position of the cutting laser. If the cuttinglaser is activated to indicate a bright spot, it is first directed awayfrom tissue or other desired or sensitive locations to avoid ablation ofwanted tissue.

The laser microdissection instrument of the present invention includesan automatic focus feature that automatically focuses the image on thetissue sample being displayed in the live video window. Focusing thetissue sample that is displayed in the live video window involves movingthe objective lens 64 in and out until the sharpest possible image ofthe subject is achieved. Depending on the distance of the subject fromthe camera, the objective has to be a certain distance from the objecttissue to obtain a clear image. The instrument may employ active orpassive autofocus techniques that well known in the art. In onevariation, an autofocus sensor such as a charge-coupled device (CCD) isincluded which provides input to algorithms and a microprocessordetermines the optimum focus distance for the objective. The instrumentautomatically focuses on the tissue when a slide is inserted into aslide location and placed in view of the acquisition system.

In addition to autofocus for the image, the laser microdissectioninstrument of the present invention further includes an automatic focuslock for the capture laser beam that advantageously keeps the capturelaser beam focused even after the objective lens is changed or thetissue being observed is varied. The capture laser beam is first focusedautomatically or manually by moving the beam to an area where there isno tissue or the tissue is very thin and light. The capture laser beamis activated and the coarse and fine focus settings are selected on thesoftware program interface and adjusted until the capture laser beam isa discrete point as viewed in the live image display window. Whenselected, the coarse and fine focus settings operate to control thelaser focus motor. The laser focus motor is connected to the controllerand computer and operates to control the focusing lens 62 and adjust thecapture laser beam focus. When not in focus, there is a large haloaround the center of the capture laser beam and the laser beam spot isblurred. When in focus, the halo around the spot is as close as possibleto the focused centroid of the laser beam. In one variation, the capturelaser focus setting is recorded as the distance of the laser focusinglens 62 to the focal plane and serves as a baseline from which thefocusing lens 62 will be moved to keep the capture laser beam in focus.

Both the objective lens and focusing lens focus on the focal plane ofthe tissue being observed. When the user navigates across the tissuesample, if necessary, the image is automatically or manually re-focusedby automatically or manually moving the objective lens up or down by anobjective distance. This objective distance is recorded. If the newlyfocused region is a region desired for capture, the instrument willprepare for capture by automatically moving the capture laser focusinglens 62 a distance from the focal plane equal to the objective distancethat the objective was moved to bring the image into focus in order tokeep the capture laser beam set at the desired focus. If the objectivelens is moved toward the worksurface on re-focus, the step ofautomatically keeping the capture laser beam at the desired focusincludes moving the laser focusing lens away from the worksurface and ifthe objective lens is moved away from the worksurface on re-focus, thestep of automatically keeping the first laser source at the desiredfocus includes moving the laser focusing lens toward the worksurface. Asthe user navigates the tissue specimen and a single objective is used,it may be necessary to refocus the image several times. Each distancethat a single objective is moved to focus the image is recorded andsummed such that, when ready for capture, the focusing lens can be keptin focus by moving the laser beam focusing lens from the baseline adistance substantially equal to the summed distance that the objectivewas moved. Alternatively, the focusing lens is moved a substantiallyequal distance from the focal plane each time the objective lens ismoved.

If a different objective lens 64 is selected on the user interface, thecomputer drives the appropriate motors to turn the objective turretwheel such that the desired objective is positioned in the primary axis.The autofocus system then immediately focuses the image based on thenewly selected objective by activating the objective focus motor to movethe objective up or down a distance to automatically adjust the foci ofthe objective which has been positioned and focus the image. However,the focusing lens is kept locked after the new objective is initiallyfocused. This distance that the objective moves to adjust for the newlypositioned objective is not recorded and summed together with the otherobjective distances. However, for any subsequent objective distancesthat the objective is moved, the focusing lens is moved accordingly, theobjective distances being recorded and summed in one variation. Hence,the capture laser is automatically focused such that the focal plane ofthe capture laser matches the focal plane of the objective lens. If theobjective lens is changed, it needs to be refocused. After it isrefocused, the focal plane of the capture laser is matched to the focalplane of the new objective.

Because the cutting laser is focused by the objective lens, the cuttinglaser beam focus is automatically maintained each time the image isfocused. If the instrument is arranged such that the cutting laser andthe capture laser locations are reversed such that the cutting laser islocated above the worksurface and the capture laser is located under theworksurface and the cutting laser is focused by the focusing lens 62 andthe capture laser is focused by the objective 64, an automatic focuslock for the cutting laser beam is provided in the same manner as thatdescribed above for the capture laser automatic focus lock. It should benoted that in one variation, both the cutting laser and the capturelaser are located on the same side of the worksurface and are bothfocused by the objective lens. In another variation, both the cuttinglaser and capture laser are located on the same side of the worksurfaceand are both focused by the focusing lens in accordance with the methoddescribed above for the capture laser automatic focus lock.

Before the capture and cutting lasers are fired, energy levels for eachare optimized according to the following methods. To optimize thecapture laser setting, the capture laser is fired with the cap inposition at an area outside desired tissue where the fired location canbe clearly observed by the user. The capture laser is fired and the areais examined for proper wetting. Wetting refers to the melting of thetransfer film on the polymer cap so that it fuses adequately to thetissue or cells when the capture laser fires. When the transfer film isvisible as a dark ring 120 fused to the slide and the center 122 of thering is clear, the wetting is adequate as shown in FIG. 11 . If thewetting is not adequate, one or more capture laser parameters such aspower and duration are adjusted. In one variation, the power setting ofthe capture laser is increased if the wetting is not adequate. Inanother variation, the duration of the capture laser is increased. Thecapture laser beam is directed at an adjacent location and test firedagain at the higher power and/or duration setting. The area is observedto determine whether wetting is adequate. If not, the process isrepeated with the power setting or duration being increasedincrementally. Determination of whether wetting is adequate may beaccomplished manually by the user via observation or automatically bythe instrument employing various algorithms. In an automated variation,the acquisition system camera image sensor records intensity data for animage of each test fire location. The microprocessor looks at thedifference in intensity among the adjacent pixels and detects a maximumintensity difference between adjacent pixels that correspond to a darkring to determine the optimum energy setting.

Referring now to FIG. 12 , in one variation, a calibration matrix isused to determine capture laser settings. The laser matrix option allowsthe user to set up a test firing pattern to determine the appropriatesettings for the capture laser to adequately wet the film. For one axisof the matrix, the user enters the number of steps for power increments,the power increment per step, and the distance between laser spots. Foranother axis of the matrix, the user enters the number of steps forduration increments, the duration increment per step, and the distancebetween laser spots. The computer automatically fires the capture lasertest pattern according to the settings to create a matrix as shown inFIG. 12 . The user then observes the pattern and selects the optimumpower and duration settings for the capture laser as desired.

To optimize the cutting laser setting, the cutting laser is fired at anarea of tissue and the area is examined for a proper tissue burn spot. Atissue burn spot typically appears white in color. If there is no burnspot detected on the tissue, one or more cutting laser parameters suchas power and duration are adjusted. In one variation, the power settingof the cutting laser is increased if the burn is not adequate. Inanother variation, the duration of the cutting laser is increased. Thecutting laser beam is directed at an adjacent location and test firedagain at the higher power and/or duration setting. The area is observedto determine whether burn sport is adequate. If not, the process isrepeated with the power setting or duration being increasedincrementally. Determination of whether the burn spot is adequate istypically accomplished manually by the user via observation.

In one variation, a calibration matrix is used to determine cuttinglaser settings as described above with respect to the capture lasercalibration matrix and FIG. 12 . The laser matrix option allows the userto set up a test firing pattern to determine the appropriate settingsfor the cutting laser to adequately cut the film. For one axis of thematrix, the user enters the number of steps for power increments, thepower increment per step, and the distance between laser spots. Foranother axis of the matrix, the user enters the number of steps forduration increments, the duration increment per step, and the distancebetween cutting laser spots. The computer automatically fires thecutting laser test pattern according to the settings to create a matrixsimilar to that shown in FIG. 12 for the capture laser. The user thenobserves the pattern and selects the optimum power and duration settingsfor the cutting laser as desired.

As described above, the instrument includes a worksurface 38 thatincludes at least one cap quality control station 54. The qualitycontrol station 54 is one or more than one location on the worksurfacedesigned for viewing the captured tissue after microdissection. Theworksurface 38 at the location of the quality control station includesan opening such that when a cap is placed in the quality controlstation, illumination or laser light passes through the opening andthrough the cap. The captured tissue adhered to the transfer film on thecap is illuminated for observation or exposed to the capture laser orcutting laser for ablation.

After capture in the slide location 46, transferred tissue is attachedto the cap and carried along therewith and placed in the quality controlstation 54. It should be noted that capture in the slide location is byany one or more of the methods described in U.S. Provisional PatentApplication Ser. No. 60/613,038, entitled “Automated microdissectioninstrument” filed on Sep. 25, 2004; U.S. patent application Ser. No.10/989,206 entitled “Automated laser capture microdissection” filed onNov. 15, 2004; U.S. patent application Ser. No. 11/222,281 entitled“Laser microdissection apparatus and method” filed on Sep. 8, 2005; andU.S. patent application Ser. No. 10/982,230 entitled “Lasermicrodissection on inverted polymer films” filed on Nov. 4, 2004 all ofwhich are incorporated herein by reference in their entirety and also asingle cap can be used to collect material from more than one tissueslide, prior to being placed in the quality control station wherefurther observation and exposure to the cutting laser and/or capturelaser is optional. For example, the sample undergoes laser capturemicrodissection with a capture laser while in the slide location priorto being placed in the quality control station where it is furtherobserved and exposed to the cutting laser and/or capture laser. Inanother example, the sample undergoes microdissection with a cuttinglaser and is exposed to the capture laser prior to being placed in thequality control station where it is further observed and exposed to thecutting laser and/or capture laser. Following cell capture in the slidelocation, the user inspects the slide and the cap in the quality controlstation to verify that collection was successful. In the quality controlstation, the camera captures a static or live image of the cap transfersurface including the accompanying captured tissue and displays it onthe computer monitor. The image is displayed either on the live videowindow or the static image window of the computer software interface,and the user inspects the quality of the capture. If there isundesirable friable tissue or other matter still attached or capture isincomplete or unsatisfactory, undesirable portions of the tissue can betargeted for ablation with the cutting laser while the cap is located inthe quality control station and/or targeted for laser capture with thecapture laser while the cap is located in the quality control station.Photoablation, the volitization of tissue by light emitted from anultraviolet cutting laser, while the cap is in the quality controlstation is performed interactively. The user ablates any unwanted tissuefrom the material on the cap. There are two modes for ablating. In thefirst mode, the user turns on the cutting laser and moves the mousecursor over the region to ablate. When finished, the user turns off thecutting laser. In the second mode, user holds one or more hot keys suchas the CONTROL and SHIFT keys at the same time and moves the mousecursor to move the laser. The cutting laser fires only while the keysare depressed and when released, the laser turns off. When inspectionand microdissection is completed in the quality control station, theuser points the mouse cursor on the cap depicted on the softwareinterface residing in the quality control station and directs the cursorto move the cap from the quality control station to the unload station52.

Referring back to FIG. 4 , a new cap, when introduced into the lasermicrodissection instrument, is located in the staging area 50 of theworksurface 38. From the staging area 50, the cap is placed on a slidelocated in any one or more slide locations 46 on the worksurface. From aparticular slide location, after microdissection for example, the capmay be moved to the quality control station or directly to a particularlocation on the unload station 52. Once the cap is moved from aparticular slide in the slide location, the cap is disassociated fromits identifying slide and tissue information. It is desirable toidentify which slide or tissue sample a cap or microdissected region isassociated with or from. To accomplish this identification, each cap istracked by recording one or more tracking information and associatingthat tracking information with each cap. A tracking information is anyuseful identification information that is associated with a particularcap. For example, the tracking information may include a cap number, capstyle, project number, or any other information. An example of a capnumber or identification is the cap's location in the cap station. Forexample, if the cap is first in the cap station it may be assigned atracking information of “CAP #1”. Additional information or notes may beassociated with this identification including date and time ofmicrodissection for example, and the style of cap that is used such asthe CapSure™ HS or CapSure™ Macro. When a cap is moved from the capstaging area 50 and into the slide location 46, it is placed on onetissue-bearing slide in a particular slide opening 48. At this point,another tracking information is recorded and associated with CAP #1. Anexample of such a tracking information is “SLIDE #3” to identify thetissue-bearing slide in a slide opening 48 on the slide location 46 onwhich the cap was placed. “SLIDE #3” for example designates the thirdslide opening 48 in the slide location 46. Additional informationassociated with a particular slide that has been entered by the user orautomatically recorded such as the slide name, tissue type, number ofcaptures, total area of captures and special notes and type of slide orany other useful or important information that is associated with “SLIDE#3” is also recorded with the other previously-recorded trackinginformations. From the slide station, the cap may be moved to thequality control station 54 and an additional tracking information suchas “QC” may be recorded and associated with the cap. If additionalablation takes place at the quality control station, that information isalso recorded and associated with the particular cap. From the qualitycontrol station, the cap is moved into the unload station 52 andpositioned in a particular location on the unload station and anothertracking information, such as the coordinates of the cap location on theunload station, “X1,Y1”, is recorded and associated with the cap. Ingeneral, as the cap is put through the steps of a prescribed procedure,a tracking information is assigned, recorded and associated with thecap. Hence, if a user wishes to track the cap it requests the trackinginformation on the user interface and a list of tracking informations orcodes associated with the cap process is outputted. For example, atracking report may include a string of tracking codes such as “CAP #1”“SLIDE #3” “EPITHELIAL” “QC” “X1,Y1”. Such a tracking report informs theuser that the captures on CAP #1 came from tissue sample on SLIDE #3.Additional tracking information that may be associated with a particularcap include a roadmap image of the slide from which tissue was captured,an information associated with a reading of a barcode located on theslide from which tissue was captured or a picture of the barcode itself.The tracking information is recorded and associated with the firstsubstrate or cap linking the first substrate or cap, the biologicalmaterial, and the at least one second substrate or tissue slide(s) fromwhich the biological material was removed. It should be noted that asingle cap can be used to collect material from more than one slide.

Alternative methods of tracking information associated with a capinclude burning a tracking code or other identified with a laser such asthe UV cutting laser onto the cap itself or onto the transfer film withthe IR capture laser in dot matrix format for example to script theinformation. Another method includes simply associating the staticroadmap image of at least a portion of the slide with the particular capsuch that the tracking information outputs the associated roadmap image.Yet another method includes, reading a slide label information such as abarcode, alpha-numeric text or other characters, and recording the slidelabel information associated with the cap. In one variation, the slidelabel information is converted and the converted data is associated withthe cap. For example, a bar code is read and converted into text datafor example and the text data is recorded and associated with the cap.Another example includes optical character recognition of characters onthe slide which is then reproduced, recorded and associated with a cap.Of course, a camera of the acquisition system can be used to captureslide information and the image itself can be associated with the cap ordata can be deciphered from the image in another variation.

All publications and patent applications mentioned in this specificationare incorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

The above description is illustrative and not restrictive. Manyvariations will be apparent to those skilled in the art upon review ofthis disclosure. The scope of the invention should not be determinedwith reference to the above description, but instead should bedetermined with reference to the appended claims and the full scope oftheir equivalents.

1. A digital microscope comprising: a worksurface for receiving asample; the worksurface intersecting a primary optical axis of themicroscope; a substrate receiving location on the worksurface forreceiving a sample-bearing substrate; the worksurface having a firstopening in the substrate receiving location for alignment with theprimary optical axis to permit pathing of light through the firstopening in the worksurface; and a digital image acquisition systemincluding an image sensor configured to: detect the presence of thesubstrate in the substrate-receiving location; and capture an image ofthe sample after the presence of a sample-bearing surface is detected inthe substrate-receiving location.
 2. The digital microscope of claim 1,wherein the digital microscope is mounted on an assembly frame andwherein the frame carries the components of the digital microscope. 3.The digital microscope of claim 2, wherein the digital microscopefurther includes an illumination system.
 4. The digital microscope ofclaim 3, wherein the illumination system comprises a white lightilluminator and a condenser mounted on the assembly frame.
 5. Thedigital microscope of claim 2, wherein the digital microscope furtherincludes a handling system.
 6. The digital microscope of claim 2,wherein the digital microscope further includes an optical system. 7.The digital microscope of claim 2, wherein the digital microscopefurther includes an illumination system and an optical system.
 8. Thedigital microscope of claim 7, wherein the illumination system,worksurface and optical system are arranged in an invertedtransmitted-light microscope fashion such that the illumination systemis arranged above the worksurface and at least one objective is arrangedbelow the worksurface.
 9. The digital microscope of claim 2, wherein theworksurface is mounted on the instrument frame and operates as atranslation stage that is automatically or manually moveable.
 10. Thedigital microscope of claim 9, wherein the worksurface moveable in alldirections including the planar X-Y directions.
 11. The digitalmicroscope of claim 1, wherein the worksurface includes slide locationsfor handling multiple tissue samples simultaneously and wherein eachslide location includes at least one opening in the worksurface.
 12. Thedigital microscope of claim 1, wherein the worksurface includes astaging area for receiving transfer film carriers and/or cap cassettes.13. The digital microscope of claim 12, wherein the worksurface furtherincludes an unload station for unloading the transfer film carriersand/or caps.
 14. The digital microscope of claim 13, wherein theworksurface further includes a quality control station, wherein thequality control station includes an opening in the worksurface thatpermits illumination and laser light to pass.
 15. The digital microscopeof claim 14, wherein the quality control station is designed for viewingthe cap following cell capture, generating an image of the cap, and/orablating portions of a collected sample residing on the transfer filmcarrier and/or cap.
 16. The digital microscope of claim 5, wherein thehandling system comprises a lift fork.
 17. The digital microscope ofclaim 16, wherein the lift fork is adapted to pick and move a transferfilm carrier and/or cap into and out of juxtaposition with thesample-bearing substrate.
 18. The digital microscope of claim 5, whereinthe handling system includes a visualization filter.
 19. The digitalmicroscope of claim 18, wherein the visualization filter comprisesdiffuser glass.
 20. The digital microscope of claim 6, wherein theoptical system includes elements mounted on the assembly frame incombination to create an optical train of optical elements for pathinglight, wherein the optical system comprises one or more optical elementsselected from a mirror, a dichroic mirror, a lens, an objective, abeam-diameter adjuster, a cut-off filter, a diffuser, a condenser, aneyepiece and an image acquisition system.